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  • Introduction CYP is a superfamily of heme containing monooxy


    Introduction CYP450 is a superfamily of heme-containing monooxygenases, many of which are expressed in the liver, and they are significant phase-I calcium ionophore in drug metabolism and detoxification. There are three subfamilies (CYP1, CYP2 and CYP3) that are mainly involved in the metabolism of drugs in both humans and rats (Nedelcheva and Gut, 1994). It has been proposed that the expression levels and activities of CYP450 enzymes directly affect the bioavailability of many drugs. Moreover, CYP450 enzymes can be inhibited, activated or induced by concomitant drug treatments, which have been recognized by regulatory authorities as an important cause of drug–drug interactions. For example, terfenadine, astemizole and cisapride are all extensively metabolized by CYP3A4 and can cause Torsades de Pointes (ventricular tachycardia) when coadministered with other drugs that inhibit CYP3A4, including ketoconazole and erythromycin (Nassar et al., 2009). Thus, the CYP450-mediated metabolism of drugs is one of the major kinetic profile determinants, and the prediction of this metabolism is highly relevant during the drug discovery and development process (Rydberg et al., 2009). Dipfluzine hydrochloride (1-diphenylmethyl-4-(3-(4-fluorobenzoyl))-piperazine hydrochloride, Dip), a diphenylpiperazine calcium channel blocker, was synthesized according to the chemical structure of cinnarizine (CZ). Previous studies have demonstrated that Dip is a highly selective cerebral vasodilator that exerts protective effects against focal or global cerebral ischemic injury via multiple mechanisms (Wang and He, 1993, Bai and Wang, 2002, Zhang et al., 2005a, Zhang et al., 2005b). In addition, studies have shown that Dip also inhibits platelet aggregation in vitro and prevents thrombus formation in vivo (Wang and He, 1994a, Wang and He, 1994b). Evidence from in vivo and in vitro experiments has revealed that the pharmacological effects of Dip are more potent than its analogs, CZ or flunarizine (FZ) (Wang and He, 1993, Zhu et al., 1996). Therefore, Dip is a promising drug candidate for the treatment of cerebral vascular diseases. A previous study showed that Dip was transformed to five metabolites via multiple pathways, mainly by N-dealkylation at the 1- and 4-positions of the piperazine ring (Liu et al., 2005). These five metabolites found in rat urine were identified by LC/DAD/MS methods as 1-(4-fluoro-benzene)-4-piperazine-butanone (M1), 4-hydroxy-benzophenone (M2), 4-fluoro-γ-hydroxy-benzenebutanoic acid (M3), diphenylmethanol (M4) and benzophenone (M5) (Fig. 1). In recent studies, we identified Dip and its metabolites using LC/MS/MS analysis in rat liver microsomes (Guo et al., 2012). The results demonstrated that only four of the five Dip metabolites found in rat urine were detected (all except M3). We presumed that the sample processing method (plasma had not been hydrolyzed) applied in our study might lead to the concentration of M3 being too low to be detected by LC/MS/MS. However, there is no evidence that the CYP450 enzymes are involved in Dip metabolism or of their relative contributions to the formation of the metabolites. Additional knowledge regarding the influence of the CYP450 enzymes on the metabolism of Dip will facilitate the development of better therapeutic strategies to enhance the efficacy and minimize the toxicity in patients. Thus, in the present study, we characterized the CYP450 enzymes involved in the metabolism of Dip using the combination of selective chemical inhibitor studies, a correlation analysis with a bank of rat liver microsomes and a panel of recombinant rat CYP450 enzymes. These results would provide useful information for future pharmacological, toxicological and clinical evaluations of Dip.
    Materials and methods
    Discussion For most drugs, biotransformation is the major route of elimination and metabolism by CYP450 enzymes is a common metabolic pathway (Rendic, 2002). Therefore, it is important to evaluate the contribution of the metabolic pathways to the elimination processes of Dip and its metabolites and to identify the CYP450 enzymes responsible for the metabolism of Dip. Thus, the present study identified and assessed the contribution of the individual CYP450 enzymes responsible for the formation of each metabolite by the combination of a chemical inhibition study, a correlation analysis and the incubation of Dip with recombinant CYP450 enzymes. Moreover, we also studied the enzyme kinetic parameters of the formation of each metabolite by rat liver microsomes and recombinant CYP450 enzymes.